Biocatalytic oxidation and reduction can be used to transform substrates with high regio- and/or stereo-selectivity. For example, biocatalytic oxidation can be used to stereospecifically hydroxylate hydrocarbons that possess no prior functional groups. Products of biocatalytic oxidation and reduction have widespread commercial application. For example, oxygenated hydrocarbons are becoming increasingly important in a range of applications, including use as blending stock for motor gasoline, intermediates for the manufacture of new less-polluting chemicals (e.g., biodegradable polymers), starting materials for synthesis of new, high-value-added products (e.g., enantiomeric pharmaceuticals), and wastewater detoxification technologies.
The cytochrome P450 enzymes are widely distributed throughout nature and participate in diversified metabolic reactions as the oxygen-activating component of monooxygenase systems, or as the dehalogenation agent of reductase systems. Their unique oxygenation chemistry and substrate specificity offer the opportunity to develop enzymatic systems for synthesizing fine chemicals, sensing biochemically active compounds, and detoxifying environmental contamination. A major hurdle to implementation of P450 catalysis for commercial syntheses is the requirement for stoichiometric amounts of freely dissociated cofactors, such as NADH (nicotinamide adenine dinucleotide hydride) and/or redox partner proteins, which supply necessary reducing equivalents. This requirement is currently met either as an additional nutrient cost during in vivo whole cell biocatalysis (fermentation) or as a cost of supplying fresh cofactor along with target substrate during in vitro biocatalysis.
A particular member of the cytochrome P450 enzymes, cytochrome CYP101 (E.C. 1.14.15.1) monooxygenase, is found in the bacterium Pseudomonas putida PpG786 when it is cultured on camphor. CYP 101 catalyzes camphor hydroxylation at the 5-exo position. Investigation has also been done on stereospecific hydroxylation of other substrates (Fruetel et al., J. Am. Chem. Soc., 114, 6987-6993 (1992); Grayson et al., Arch. Biochem Biophys., 332, 239-247 (1996)) as well as dehalogenation reactions (Koe & Vilker, biotechnology Progress, 9, 608-614 (1993)).
In its normal physiological role (i.e., natural cycle), this three-protein enzyme system hydroxylates camphor using NADH as the source of reducing equivalents. The reactions require NADH as well as two protein cofactors. The overall biocatalytic cycle involves many individual reactions. The two electrons necessary for the conversion of the substrate are supplied by reduced putidaredoxin (Pdx), a 2Fe-2S protein, which mediates the transfer of the electrons from NADH, and the FAD-containing putidaredoxin reductase (PdR) to the heme active center of the cytochrome CYP101. In addition to being an electron mediator, Pdx is also an "effector" for product release in the final step of the cycle. The overall reaction is thermodynamically controlled, and redox potential and substrate binding are modulated by the cytochrome spin-state equilibrium.
In industrial applications, NADH is lost by decomposition and must be replenished to continue the reaction. NADH is expensive, however, and thus economic feasibility requires that simple, effective and stable methods for cofactor recycling be found. Electrochemical cofactor regeneration can meet these requirements.
Some attempts have been made to use electrodes to supply the reducing power for driving P450 catalytic cycles. Bioelectrochemical processes have been described in which electrons are transferred directly (without mediators) between an electrode and redox-active biological material, such as an enzyme or protein, using various modified metal or graphite electrodes. Such processes suffer, however, from either inefficiency (low redox reaction rates) or rapid decline in activity due to component fouling by proteins.
Higgens et al. (U.S. Pat. No. 4,318,784) and Armstrong (Structure and Bonding, 72, 137-221, 1990) describe using metal and graphite electrodes modified with organic absorbates for the electron transfer to and from various redox-active biomolecules. These systems are inefficient and/or unstable, however, when applied to hydroxylation of organic compounds by the P450 cycle. Some of the problems with these systems include (i) modifier instability in the required potential range, (ii) irreversible adsorption of protein constituents leading to electrode fouling, and (iii) protein denaturation. See also Kazlauskaite et al. (Chem. Commun., 2189-2190, 1996) and Zhang et al. (J. Chem. Soc. Faraday Trans., 93, 1769-1774, 1997), which describe direct electron transfer from carbon-based electrodes to CYP101. The direct electron transfer study of Kazlauskaite et al. showed reversible electrochemical response from glassy carbon electrodes, corresponding to the first of the two electron transfers to CYP101 required to initiate the catalytic cycle, while Zhang et al. showed direct electron transfer from lipid-modified pyrolytic graphite electrodes to CYP101. Neither Kazlauskaite nor Zhang demonstrated oxygenase enzyme activity.
Faulkner et al. (Proc. Natl. Acad. Sci. USA, 92, 7705-7709, 1995) have shown mediated, electrode-driven biocatalysis using cobalt (III) sepulchrate to transfer electrons to rat recombinant liver P450 fusion proteins. In these mediated biocatalysis studies, the electrolysis enzyme turnover rate was comparable with the NADPH-driven cycle for a number of recombinant fusion microsomal P450 enzymes. See also Estabrook et al. (Methods in Enzymology, Cytochrome P450, Part B, eds. Johnson & Waterman, 44-50, 1996). The drawback to this method is the use of the cobalt mediator, which is expensive and difficult to remove from the reactants and products.
Previous investigation was done on the use of polycrystalline gold and silver electrodes modified by immobilizing ionizable organic molecules in order to overcome the repulsive electrostatic interaction that arises when trying to reduce the negatively-charged Pdx at the electrode. Reversible oxidation/treduction cycling of Pdx on these electrodes was observed, but the response was short-lived. Both in situ, surface-enhanced Raman spectroscopic and ellipsometric measurements demonstrated rapid deterioration of the organic electrode modifier while the electrode was held at potentials less than -0.7 V.
A clear need exists for an electrode that can rapidly and continuously reduce Pdx while having a minimal effect on other reaction components. The successful electrode must not be irreversibly coated by other components, including CYP101. Additionally, in order for the cytochrome P450 to participate repeatedly as a catalyst in hydroxylation reactions, it must be provided continually with oxygen. However, oxygen plays a dual role in these reactions, acting as a co-reactant for product formation and oxidizing the reduced components, thus decreasing the turnover of reactant into product. Previously, oxygen was supplied to such reactions by contacting the reactor solution with atmospheric air, or by purging the reaction solution with pure oxygen gas. These methods are disadvantageous, however, because they increase the oxygen concentration so high as to stop the reaction at the initial stages (by taking up electrons from the working electrode or reduced reactants). As a result, the product yield is low. Thus, a need exists for a new method for supplying oxygen to the reaction that results in a higher product yield.